Our long-term objective is to understand the common and distinct pathways, mechanisms and regulation of membrane traffic in polarized epithelial cells. Current evidence suggests that there may be different delivery pathways, sorting locations and dependencies on microtubules of membrane traffic in the different epithelial cells, as represented by kidney, intestine and liver. We will conduct a careful comparative study of vesicle traffic in these three epithelial cell types using in vitro model systems. For kidney, we will use the MDCK cell; for intestine, the Caco-2 cell; and for liver, a newly-generated rat hepatocyte/human fibroblast hybrid called WIF12-1. We will first determine the extent to which the WIF12-1 cells resemble polarized hepatocytes in situ, by: 1) examining the steady-state distributions of domain-specific membrane proteins using immunofluorescence (IMF) and improved immuno-electron microscopic techniques; and 2) determining the kinetics of and intracellular compartments involved in secretion, transcytosis using metabolic precursors (amino acids) and labeled ligands (e.g., 125I-polymeric IgA 125I- asialoglycoproteins, horseradish peroxidase-ligands). With Doug Fambrough (Project #2), who will be generating MDCK, Caco-2 and (presumably) WIF12-1 cells that have been transfected with native and chimeric cDNA constructs of apical (DPPIV), basolateral (Na,K-ATPase) and lysosomal (LEP100) membrane proteins and an unsorted secretory protein (kappa light chain), we will compare the pathways and compartments involved in the sorting and delivery of these molecules to their destinations. We will use metabolic labeling (amino acids, carbohydrates), cell surface probers (e.g., chemical labels, proteases, antibodies) in conjunction with immunoprecipitation and SDS-PAGE/fluorographic analysis in kinetic experiments and IMF/immuno-EM for steady-state distributional analyses. With Dick Pagano (Project #5) and Trina Schroer (Project #6), we will use selective perturbations to study the regulation of membrane traffic in cells transfected with constructs judged interesting from Aim #2. The perturbations are: 1) cholesterol depletion by growth in lipoprotein-deficient serum; 2) inhibition of sphingo-glycolipid metabolism using a new inhibitor, PDMP; and 3) microtubule disruption. All three appear to perturb membrane traffic at the Golgi. The approaches will include immuno-morphology and kinetic experiments. Finally, we will initiate a search for the molecular determinants of hepatocyte polarity using the WIF12-1 cells, their parental lines and cDNA substraction methods.